Figure 3.

Identification of a gene encoding bile acid 12β-HSDH. (a) SDS-PAGE of candidate Clostridium paraputrificum 12β-HSDH proteins that were heterologously expressed in E. coli and purified with TALON® metal affinity resin. Lanes are as follows: S, molecular weight protein standard; 077, WP_027099077.1; 355, WP_027098355.1; 937, WP_027097937.1; 604, WP_027098604.1; 909, WP_027096909.1; 631, WP_027099631.1. (b) Representative negative ion mode LC-MS chromatograms in single ion monitoring mode overlaid with linked vertical axes of WP_027099077.1 reaction products compared to deoxycholic acid (DCA), 12-oxolithocholic acid (12-oxoLCA) and epiDCA standards. Standards were run on a separate day and show a slight offset in elution time. Reactions consisted of 10 nM WP_027099077.1 with 50 μM (or no) substrate, 150 μM pyridine nucleotide in 50 mM sodium phosphate, 150 mM sodium chloride buffer at pH 7.0. Formula weight for DCA is 392.57 atomic mass units (amu), 12-oxoLCA is 390.56 amu, epiDCA is 392.57 amu